In this collaborative project, the biochemistry and molecular biology of Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase (TeSADH) will be investigated. This enzyme has a novel metabolic biochemistry, since physiologically it synthesizes ethanol in two reductive steps from acetyl-CoA. This, in part, explains its natural broad substrate specificity. In this project, TeSADH will be used as a model system to understand the physicochemical bases for substrate specificity and stereospecificity of enzymes. Genetic engineering studies of TeSADH will include: 1) Rational design of the small substrate-binding pocket to determine its role in (S)- versus (R)-enantiospecificity; 2) Evolving TeSADH by directed evolution from an (S)-specific enzyme into an enzyme with high (R)-enantiospecificity; and 3) Changing TeSADH cofactor specificity from NADP+ towards NAD+. TeSADH is uniquely suited for these investigations because of its high thermostability and its broad substrate specificity. The effects of the mutations will be evaluated in terms of substrate specificity, kinetic parameters, and enantiomeric specificity.
Broader Impacts: The modified enzymes resulting from this research could be potentially used in preparation of chiral intermediates in commercial synthesis. The results of the proposed research are likely to make a significant intellectual contribution to a greater understanding or the physical biochemical basis of enzymatic stereospecificity, as well as to provide novel biocatalysts with potential industrial utility. The project's outreach program includes training multidisciplinary graduate and undergraduate students, including minorities; disseminating knowledge in courses at the grantee institutions; and presenting papers at national chemistry, biochemistry, chemical engineering, and microbiology meetings.
